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Calcium accelerates endocytosis of vSNAREs at hippocampal synapses

Nature Neuroscience volume 4, pages 129–136 (2001)Cite this article

Abstract

A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.

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Figure 1: The rate of synapto-pHluorin clearance from presynaptic membranes slows in lower [Ca2+]ex.
Figure 2: 'Alkaline trapping' to measure exocytosis and endocytosis at synaptic terminals.
Figure 3: Bafilomycin prevents acidification of newly endocytosed vesicles without slowing endocytosis.
Figure 4: Endocytosis speed is constant during and immediately following the stimulus.
Figure 5: Calcium controls the speed of endocytosis.

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Acknowledgements

We thank J. Rothman for providing the super-ecliptic synapto-pHluorin construct, T. McGraw, F. Maxfield and D. Eleizer for discussions, and W. Yan for technical assistance. This work was supported by the NIH (NS24692) and a McKnight Technological Innovations in Neuroscience Award to T.A.R. T.A.R. is an Alfred P. Sloan Research fellow.

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  1. Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Ave, New York, 10021, New York, USA

    Sethuraman Sankaranarayanan & Timothy A. Ryan

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  2. Timothy A. Ryan
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Correspondence to Timothy A. Ryan.

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Sankaranarayanan, S., Ryan, T. Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nat Neurosci 4, 129–136 (2001). https://doi.org/10.1038/83949

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